Contrast medium for near infrared diagnosis

ABSTRACT

This invention relates to colloidal systems charged with polymethine dyes and having suitable photophysical and pharmacological properties, their use as a contrast medium in fluorescence and transillumination diagnostics in the near infrared spectral range, as well as methods for their production.

This application is a 371 of PCT/DE96/01878 filed Sep. 26, 1996.

FIELD OF THE INVENTION

This invention relates to colloidal systems charged with polymethine dyes, their use as a contrast medium in fluorescence and transillumination diagnostics in the near infrared spectral range, as well as methods for their production.

BACKGROUND OF THE INVENTION

As biological tissue has relatively high permeability for long-wave light in the range of 700 to 1000 nm, a diagnostician can therefore use a completely different method of tissue imaging in addition to advanced medical imaging techniques such as X-raying, magnetic resonance tomography, or ultrasonic diagnosis.

Tissue-specific information can be obtained both by detecting the non-absorbed portion of radiation by means of a transmission image, and by detecting fluorescence radiation emitted after exposing the tissue to light in the near infrared range.

The main problem with the use of near infrared radiation is the extraordinarily wide scattering of light, which yields only a rather blurred image of a clearly contoured object despite different photophysical properties of such an object and its environment. This problem intensifies the greater the distance from the surface and may be considered the major limiting factor of both transillumination and the detection of fluorescence radiation.

SUMMARY OF THE INVENTION

Suitable fluorescent dyes that accumulate in diseased tissue (particularly in tumours) and display a specific absorption and emission behaviour may contribute towards enhancing the distinction of healthy from diseased tissue. The change in the irradiated (scattered) light caused by absorbtion of the dye, or fluorescence induced by exciting radiation, is detected and provides the actual tissue-specific information.

Until now, photosensitizers designed for use in photodynamic therapy (PDT) (including porphyrins, chlorines, phthalocyanines, naphthalocyanines) have been used for localizing and visualizing tumours (Bonnett R. New photosensitizers for the photodynamic therapy of tumours. SPIE Vol. 2078, 1994). The classes of compounds listed here share the disadvantage that in the wavelength range of 600 to 1200 nm they are either not absorbing at all or to a very little extent only. The photosensitizing effect of these dyes is disturbing for purely diagnostic uses where no effects are desired. Furthermore, photostability of the dyes listed here often is quite low.

In contrast to this, the absorption and fluorescent behaviour of dyes from the class of polymethines is characterized by high absorption coefficients in the range between 700 and 1000 nm and a sufficient fluorescent quantum yield. The photosensitizing effect of polymethines can be neglected, and most of them are highly photostable.

A clear change in the pharmacokinetic properties of fluorescent dyes may be achieved by transforming them into colloidal systems. As a result, a tissue or organ-specific, or location-specific accumulation of the fluorescent dyes formulated in this way may be achieved.

Furthermore, the dyes must be strongly hydrophilic to be applied in an aqueous solution so that sufficient quantities of dye for imaging can be introduced into the body using a water solution.

It is known that the transformation of dyes into colloidal systems may increase the applicable dose.

It is an object of this invention to provide a contrast medium that accumulates to a considerable extent in the tissues to be examined and can be detected in said tissues using near infrared radiation.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts fluoroscopic imaging of a nude mouse with an LS174T tumour after application of a contrast medium according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This problem is solved according to the invention by providing contrast media containing colloidal systems charged with dye, said colloids having sizes from 5 nm to 10 μm and containing a minimum of one dye that absorbs and/or fluoresces in a wavelength range from 600 to 1200 nm. For a definition of colloids, see Hunnius, Pharmazeutisches Wörterbuch, 6th edition, Berlin: de Gruyter 1986, pp. 589 f.; this description, however, includes coarsely dispersed systems with particle sizes up to 10 μm in the concept of colloidal systems.

Surprisingly, it was found that colloidal systems charged with dye accumulate in local inflammation or tumor areas. This accumulation is excellently suited for visualizing tumours or inflammations using NIR diagnosis.

Furthermore, it was surprisingly found that the contrast media of the invention are quickly degraded in specific cells, for example, in Kupfer cells of the liver, which results in a rapid decrease of colloid concentrations in the blood, while the colloid particles already absorbed by the target tissue are degraded at a much slower pace. This reduces the background noise caused by the concentration of contrast medium in the blood, which improves the visualization of lesions and/or makes them visible at an earlier stage.

This invention thus relates to colloid systems charged with dye and containing colloidal particles of sizes between 5 nm and 10 μm with dye molecules integrated in, adhering to, or encapsuled by their walls and/or cavities.

Suited are all systems in which the dyes are integrated in colloidal particles consisting of biodegradable, partially synthetic materials or materials identical with natural substances. The properties and methods of preparing suitable colloidal particles are described, for example, in Boyett, J. B., and Davis, C. W. Injectable Emulsions and Suspensions. In: Liebermann, H. A., M. M. Rieger, and G. S. Banker, eds. Pharmaceutical Dosage Forms: Disperse Systems. Vol. 2. New York: Marcel Dekker, 1988, 379-416.

Particularly suitable are systems in which the synthetic polymeric material has been selected from the following group: Poly-ε-caprolactone, polylactic acid, polyglycolic acid, and mixed polymers from polylactic and polyglycolic acid, polyhydroxy butyric acid, polyhydroxy valeric acid, and mixed polymers from polyhydroxy butyric acid and polyhydroxy valeric acid, polyamino acids, polyalkyl cyanoacrylates, polyamides, polyacrylodextrane, polyacrylic starch, polyacrylosaccharide, polyacrylamide, polyester, poly(ortho)ester, polyphosphorenes, and copolymers of lactic acid and/or glycolic acid and polyoxyethylene. Preferred natural or partially synthetic biodegradable polymeric materials primarily include proteins such as albumins, collagen, gelatin, haemoglobin or fibrinogen and starches, dextranes, chitin and chitosan.

Particularly suitable are also amphiphilic substances that either encapsule the fluorescent dye in colloidal particles or form colloidal particles together with the fluorescent dye. Preferred substances are phospholipids, fatty acids, fatty alcohols, cholesterol, esters or ethers of fatty alcohols or fatty alcohols and fatty acids, sugar derivatives with fatty acids or polyoxyethylene, esters or ethers of phospholipids, fatty acids, fatty alcohols with polyoxyethylene, bile acids, derivatives of sorbitan with polyoxyethylene or fatty acids or fatty alcohols as well as their combinations.

Preferred contrast media according to the invention contain colloids from proteins such as albumins, collagen, gelatin, haemoglobin or fibrinogen, or starches and starch derivatives, dextranes, chitin, or chitosan.

Other preferred contrast media according to the invention contain colloids from phospholipids, fatty acids, fatty alcohols, cholesterol, esters of fatty alcohols and fatty acids, ethers of fatty alcohols and fatty acids, sugar derivatives containing fatty acids or polyoxyethylene, esters or ethers of phospholipids, fatty acids or fatty alcohols with polyoxyethylene, bile acids, derivatives of sorbitan with polyoxyethylene, fatty acids or fatty alcohols, and combinations of said substances.

Particularly preferred are such contrast media of the invention that contain polyesters of α-, β-, γ- or ε-hydroxycarboxylic acids, polyalkyl cyanoacrylates, polyamino acids, polyamides, polyacrylated saccharides or poly(ortho)esters in the form of colloids.

The dyes used for the colloidal near-infrared diagnostic agent are characterized by the fact that they absorb and fluoresce in a wavelength range of 600 to 1200 nm, have absorption coefficients of about 100,000 l mol⁻¹ cm⁻¹ and higher, and, inasmuch as fluorescence is desirable, show fluorescence quantum yields of more than 5%.

The dyes used are of the class of polymethine dyes selected from the following group: cyanine, styryl, merocyanine, squaraine, and oxonol dyes.

Preferred are dyes of the class of cyanine dyes having maximum absorption and fluorescence values between 700 and 1000 nm and extinction coefficients of about 140,000 l mol⁻¹ cm⁻¹ and more, and carrying one or several unsubstituted, branched or non-branched, acyclic or cyclic or, optionally, aromatic carbon-hydrogen residues and/or containing oxygen, sulfur, nitrogen.

The contrast media of the invention contain a cyanine, styryl, merocyanine, squaraine, or oxonol dye, or a mixture of said dyes.

Preferred are contrast media according to the invention that contain a dye from the class of cyanine or squaraine dyes.

Also preferred are contrast media according to the invention in which the dye carries one or several additional, branched or non-branched, cyclic or polycyclic alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, aryl, alkylaryl or arylalkyl residue(s), each containing up to 60 carbon atoms, and optionally carrying additional halogen atoms, hydroxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, oxo, oxy, or alkoxy groups that contain up to 20 carbon atoms, and/or may be interrupted and/or replaced by one or several heteroatoms of the O, N, S, or P series.

Particularly preferred are contrast media of the invention that contain at least one dye of the general formula I, II, or III

wherein

R¹, R², R³, R⁴, R⁷, R⁸, R⁹, and R¹⁰ are the same or different, and each of which independently represents a —COOE¹, —CONE¹E², —NHCOE¹, —NHCONHE¹, —NE¹WE², —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E², —E¹ residue, a fluorine, chlorine, bromine, or iodine atom or a nitro group, or where 5- to 6-membered rings are anellated to each pair of adjacent residues R¹, R², R³, or R⁴, R⁷, R⁸, R⁹ or R¹⁰ respectively, depending on the C atoms located in between, said rings being either saturated or unsaturated or aromatic and optionally carrying additional —COOE¹, —CONE¹E², —NHCOE¹, —NHCONHE¹, —NE¹E², —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E² residues,

wherein

E¹ and E² are same or different, each of which independently represents a hydrogen atom, a saturated or unsaturated, branched or non-branched C₁-C₅₀ alkyl chain, and said chain or parts thereof optionally forms a cyclic C₅-C₆, or a bicyclic C₁₀ unit interrupted and/or replaced by oxygen atoms, sulfur atoms, nitrogen atoms, carboxylic acid ester, carboxylic acid amide, urea, thiourea, carbamate or ether groupings, or represent a hydroxypolyoxyethylene or methoxypolyoxyethylene chain or a branched or non-branched C₁-C₁₀ alkyl chain substituted with 1 to 19 fluorine atoms,

R⁵ and R⁶ independently represent an —E¹ residue or a C₁-C₄ sulfoalkyl chain,

where E1 is as defined above

Q represents a

fragment

wherein

R¹¹ is a hydrogen, fluorine, chlorine, bromine, or iodine atom, a —NE¹E², —OE¹, or —E¹ residue or a nitro group,

R¹² represents a hydrogen atom or an —E¹ residue,

b is one of the numbers 0, 2 or 3,

where E¹ and E² are as defined above,

X and Y are same or different, each of which independently represents O, S, —CH═CH—, or a

fragment,

wherein

R¹³ and R¹⁴ independently represent hydrogen, a saturated or unsaturated, branched or non-branched C₁-C₁₅ alkyl chain, and where the residues R¹³ and R¹⁴ may be interconnected by forming a 5- or 6-membered ring, said members being interrupted and/or replaced by oxygen atoms and/or hydroxy groups, alkoxy groups containing up to 6 carbon atoms, carboxylic acid ester and/or carboxylic acid amide units,

or their carboxylic acid and/or sulfonyl salts with physiologically tolerable inorganic or organic cations.

Sodium, potassium, calcium, magnesium, gadolinium, and lysine, glutamine and methyl glutamine may occur as cations.

Dyes with a positive total charge are preferably iodides, bromides, or perchlorates.

The contrast media according to the invention may furthermore contain a minimum of two dyes having different photophysical and/or pharmacological properties.

The contrast media according to the invention may furthermore contain adjuvants, substrates, and diluents common in galenics.

The dyes are prepared using methods known from the relevant literature as, for example, Hamer, F. M. The Cyanine Dyes and Related Compounds. New York: John Wiley and Sons, 1964; Bioconjugate Chem. 4 (1993), 105-11; Anal. Biochem. 217 (1994), 197-204; Tetrahedron 45 (1989), 4845-66; Anal. Chim. Acta 282 (1993), 633-641; Dyes Pigm. 21 (1993), 227-234; EP 0 591 820 A1.

Another object of this invention is the use of the contrast media of the invention for in vivo fluorescence and absorption diagnosis in the near infrared range.

In vivo diagnosis using the contrast media of the invention is preferably carried out after intravenous administration of the substances by irradiating with monochrome light of a wavelength range of 600 to 1200 nm and position-dependent detection of non-absorbed radiation and/or fluorescent radiation. A synthetic image is generated based on the data obtained.

The diagnostic agent of the invention is prepared in analogy with the methods for the production of colloidal particles described in the relevant literature in which one or several dye(s) is/are added to the reaction mixture. The charging ratio can be varied by setting the reaction system to another dye concentration.

The preferred method is to dissolve the organic materials described above together with one or more dyes in one or several organic solvent(s) that cannot be mixed with water, and to emulgate them after optionally adding another solvent, an emulsifying agent may optionally be added to the emulsion. The colloidal system obtained can subsequently be filtered and stabilized by drying, e.g. by lyophilizing.

These contrast media are also manufactured according to methods known to an expert skilled in the art which may involve the use of common adjuvants and/or diluents, and the like. This includes physiologically tolerable electrolytes, buffers, detergents, emulgators, and substances used to adjust osmolality or to enhance the stability and solubility of the contrast media, e.g. cyclodextrines. The sterility of the preparations during the manufacturing process and, above all, when applying them, is to be ensured by common pharmaceutical measures.

The following examples shall explain the invention in some more detail.

EXAMPLE 1 Production of a Particle Suspension Containing 1,1′,3,3,3′,3′-hexamethyl Indotricarbocyanine Iodide

7.6 mg of hexamethyl indotricarbocyanine iodide and 0.2 g of a copolymer of lactic acid and glycolic acid having a molecular mass of about 15,000 g/mol are dissolved in 2.5 ml of methylene chloride. This solution is added under vigorous stirring to 20 ml of a 2% solution of gelatin that has been autoclaved for 15 minutes at 121° C. The mixture is kept agitated for another 45 minutes. The suspension thus obtained is filled into 20 ml glass vessels in portions of 5 ml and frozen directly using liquid nitrogen. Subsequently, the frozen suspension is lyophilized. After re-suspending a portion with 5 ml of a 0.9% solution of common salt, the suspension contains about 10¹⁰ particles containing hexamethyl indotricarbocyanine iodide per ml with particle sizes from about 1 to 10 μm.

EXAMPLE 2 Application of the Preparation from Example 1

Fluoroscopic imaging of a nude mouse with a LS174T tumour after applying 200 μl of a poly-(1-lactide-glycolide) particle suspension (10¹⁰ particles/ml) with encapsuled hexamethyl indotricarbocyanine iodide (0.6 μmol/ml) using a CCD camera yielded images showing fluorescence mainly in the tumour tissue after 24 hours (FIG. 1). 

What is claimed is:
 1. An in vivo contrast medium and target tissue combination, wherein said target tissue is selected from the group consisting of inflamed tissue and tumor tissue, said in vivo contrast medium comprising a colloidal system with particles having a size range from 5 nm to 10 μm, said colloidal system further comprising at least one dye fluorescing in the wavelength range from 600 nm to 1200 nm upon direct absorption of monochromatic light in the wavelength range from 600 nm to 1200 nm, said at least one dye is selected from the group consisting of the general formula I,

wherein R¹-R¹⁰ represent the residue E¹, where E¹ is a hydrogen, wherein Q is one of the following fragments:

wherein b=0, R¹¹ represents a hydrogen atom, R¹² represents a hydrogen atom or a saturated or unsaturated, branched or non-branched C₁-C₅₀ alkyl chain, and said chain or parts thereof form a cyclic C₅-C₆, or a bicyclic C₁₀ unit interrupted and/or replaced by oxygen atoms, sulfur atoms, nitrogen atoms, carboxylic acid ester, carboxylic acid amide, urea, thiourea, carbamate, ether groupings, or represent a hydroxypolyoxyethylene or methoxypolyoxyethylene chain or a branched or non-branched C₁-C₁₀ alkyl chain substituted with 1 to 19 fluorine atoms, where X and Y are both

fragment, and wherein R¹³ and R¹⁴ independently represent hydrogen.
 2. The contrast medium according to claim 1, characterized in that the colloids contain proteins such as albumins, collagen, gelatin, haemoglobin or fibrinogen, or starches, dextranes, chitin or chitosan.
 3. The contrast medium according to claim 1, characterized in that the colloids contain phospholipids, fatty acids, fatty alcohols, cholesterol, esters of fatty alcohols and fatty acids, ethers of fatty alcohols, sugar derivatives with fatty acids or polyoxyethylene, esters or ethers of phospholipids, fatty acids, or fatty alcohols with polyoxyethylene, bile acids, derivatives of sorbitan with polyoxyethylene, fatty acids, or fatty alcohols, as well as combinations of said substances.
 4. The in vivo contrast medium according to claim 1, wherein said at least one dye further comprises at least two dyes of general formula I.
 5. The in vivo contrast medium according to claim 1, wherein said in vivo contrast medium farther comprises at least one adjuvant, at least one substrate, and at least one diluent common in galenics.
 6. A method for in vivo near infrared diagnosis of target tissue in an organism using an in vivo contrast medium capable of absorption and/or fluorescence in the 600 nm to 1200 nm wavelength range, said target tissue is selected from the group consisting of inflamed tissue and tumor tissue, said method comprising the steps of: (a) preparing an in vivo contrast medium comprising a colloidal system, said colloidal system having a particle size range from 5 nm to 10 μm, said colloidal system comprising at least one dye, said at least one dye absorbs and/or fluoresces in the wavelength range from 600 nm to 1200 nm, said in vivo contrast medium being amenable to accumulation in said target tissue, said in vivo contrast medium being amenable to degradation by liver tissue at a faster rate than degradation by said target tissue; (b) performing intravenous administration of said in vivo contrast medium into an organism; (c) sufficiently irradiating said organism with monochrome light of a wavelength range of about 600 nm to 1200 nm to cause said in vivo contrast medium to fluoresce; and (d) scanning said irradiated organism using a CCD camera to obtain images of said in vivo contrast medium to detect the diseased tissue.
 7. The method of claim 6 further comprising repeating steps (c) and (d) about 10 minutes after step (a), 1 hour after step (a), 18 hours after step (a), and 24 hours after step (a).
 8. An in vivo contrast medium and target tissue combination, wherein said target tissue is selected from the group consisting of inflamed tissue and tumor tissue, said in vivo contrast medium comprising a colloidal system, said colloidal system having a particle size range from 5 nm to 10 μm, said colloidal system comprising a dye, said dye absorbing and/or fluorescing in the wavelength range from 600 nm to 1200 nm, said dye is 1,1′,3,3,3′,3′-hexamethyl indotricarbocyanine iodide of Example
 1. 9. A method for imaging tumor tissue comprising the steps of: (a) performing intravenous administration of an in vivo contrast medium into a body suspected of harboring tumor tissue, said in vivo contrast medium comprising colloidal system with a particle size range from 5 nm to 10 μm, said colloidal system comprising one or more dyes, said one or more dyes absorbing and/or fluorescing in the wavelength range from 600 nm to 1200 nm, said in vivo contrast medium being amenable to accumulation in said tumor tissue, said in vivo contrast medium being amenable to degradation by liver tissue at a faster rate than degradation by said tumor tissue; (b) irradiating said body with monochrome light in the wavelength range of about 600 nm to 1200 nm to cause said in vivo contrast medium of step (b) to absorb and/or fluoresce; and (c) scanning said irradiated body to yield images of said tumor tissue in said body.
 10. The method of claim 9, wherein step (c) further comprises using a CCD camera to provide images of said tumor tissue.
 11. The method of claim 9, wherein said body is selected from the group comprising of a human patient and an animal in possession of a liver. 